Directly induced hepatogenic cells derived from human fibroblast ameliorate liver fibrosis.

Recently, reprogramming technology has emerged as a fascinating tool to generate specific tissue cells. In this study, we tested the hypothesis that ultrasound-directed cellular reprogramming can generate fibroblasts into hepatogenic cells. We directly induced human dermal fibroblasts (HDFs) into hepatocyte-like cells mediated by environmental transition-guided cellular reprogramming (h/entr) using ultrasound. We confirmed the characteristics of h/entr by the expression levels of hepatocyte specific RNA and proteins. The effects of h/entr on the activation of hepatic stellate cells were analyzed using conditioned medium (CM). h/entr were transplanted into mice with acute liver fibrosis and the therapeutic effects and mechanism of liver fibrosis were determined. h/entr exhibited high levels of hepatocyte specific genes, hepatogenic (hepatocyte growth factor [HGF], colony-stimulating factor 3 [CSF-3]) and anti-inflammatory (interleukin 10 [IL-10]) factors. h/entr CM suppressed the activation of hepatic stellate cells in vitro. Transplantation of h/entr significantly delayed liver fibrosis and improved liver function. Transplantation of h/entr accelerates liver regeneration, and human albumin expressing h/entr and human Alu gene were detected in the mouse livers. This report suggests that directly induced h/entr could be one of the highly effective therapeutic options for the treatment of liver cirrhotic disease.

Transplantation of human adipose tissue derived-SVF enhance liver function through high anti-inflammatory property.

Although human adipose tissue-derived stromal vascular fraction (SVF) has been considered a promising source of stem cells, its characteristics relevant to treatment of a damaged liver have not been fully elucidated. In the present study, we sought to characterize the property of human SVF and determine the therapeutic utility of SVF in the liver cirrhosis model. We performed microarray, quantitative (q)-PCR experiments, and in vivo therapeutic assays using a liver cirrhotic mouse model. q-PCR results revealed that hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF)-A, Interleukin (IL)-10 and microRNA (miR)-146 were more highly upregulated in SVF than in human adipose-derived mesenchymal stem cells (ASCs). The SVF culture medium (CM) inhibited the activation of hepatic stellate cells in vitro. Injection of SVF significantly suppressed TAA-induced liver fibrosis and repaired liver function by inhibition of infiltrating inflammatory cells and induction of capillary/hepatocyte regeneration in vivo. Injection of IL-10 siRNA treated SVF cells decreased anti-inflammation and anti-fibrotic effects in TAA-induced mice liver. Our data indicate that SVF show a high anti-inflammatory property for treating fibrotic liver diseases through IL-10 secretion. Therefore, SVF might be a novel therapeutic alternative for the treatment of liver cirrhosis in clinical settings.

Cytotoxic effects of ex vivo-expanded natural killer cell-enriched lymphocytes (MYJ1633) against liver cancer.

BACKGROUND: Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a targeted method of controlling the immune system against cancer. Despite their significant therapeutic potential, efficient methods to generate adequate numbers of NK cells are lacking and ex vivo-expansion and activation of NK cells is currently under intensive investigation. The primary purpose of this study was to develop an effective method for expansion and activation of the effector cells with high proportion of NK cells and increasing cytotoxicity against liver cancer in a short time period. METHODS: Expanded NK cell-enriched lymphocytes (NKL) designated as "MYJ1633" were prepared by using autologous human plasma, cytokines (IL-2, IL-12 and IL-18) and agonistic antibodies (CD16, CD56 and NKp46) without an NK cell-sorting step. The characteristics of NKL were compared to those of freshly isolated PBMCs. In addition, the cytotoxic effect of the NKL on liver cancer cell was examined in vitro and in vivo. RESULTS: The total cell number after ex vivo-expansion increased about 140-fold compared to that of freshly isolated PBMC within 2 weeks. Approximately 78% of the expanded and activated NKL using the house-developed protocol was NK cell and NKT cells even without a NK cell-sorting step. In addition, the expanded and activated NKL demonstrated potent cytotoxicity against liver cancer in vitro and in vivo. CONCLUSION: The house-developed method can be a new and effective strategy to prepare clinically applicable NKL for autologous NK cell-based anti-tumor immunotherapy.

Human Adipose Derived Stem Cells Exhibit Enhanced Liver Regeneration in Acute Liver Injury by Controlled Releasing Hepatocyte Growth Factor.

BACKGROUND/AIMS: Although mesenchymal stem cells (MSCs) provide effective therapy for liver fibrosis, there are conflicting data regarding their marginal therapeutic effects. This study aimed to enhance the potential of hepatocyte regeneration in human adipose mesenchymal stem cells (ASCs) and investigate whether they have robust therapeutic efficacy in experimental liver fibrosis. METHODS: ASCs were cultured with four cytokines (ASC-C), the expression of hepatogenic factors was detected by microarray, and the effects of conditioned medium (CM) from ASC-C on the activation of hepatic stellate cells were analyzed. The therapeutic effects and mechanism of liver fibrosis induced by thioacetamide (TAA) were determined after cell transplantation. RESULTS: ASC-C exhibited high levels of hepatogenic (HGF, G-CSF), anti-apoptotic (IGFBP-2), and chemokine (IL-8) genes and increased expression of hepatocyte specific proteins. ASC-C CM inhibited the activation of hepatic stellate cells in vitro, and injection of ASC-C significantly delayed TAA-induced liver fibrosis and improved liver function and regeneration in vivo. In addition, human albumin-expressing ASC-C were observed in the livers of recipient animals. High levels of expression of HGF and its downstream signaling molecules, including p-38, were detected in the ASC-C-injected livers. Transplantation of ASC-C exerts anti-fibrotic effects and accelerates liver regeneration. CONCLUSION: Thus, ASC-C may be a novel candidate for the enhanced treatment of liver cirrhosis in clinical settings.

Generation of directly reprogrammed human endothelial cells derived from fibroblast using ultrasound.

Physical microenvironment plays an important role in determining cellular reprogramming. In this study, we first generated directly reprogrammed human dermal fibroblasts (HDFs) into endothelial cells (ECs) mediated by environmental transition-guided cellular reprogramming (e/Entr) using ultrasound and characterized e/Entr. Ultrasound stimulus was introduced to ECs culture media and HDFs and induced into ECs-like cells. We performed microarray, RT-PCR, protein analysis, matrigel plug assay and e/Entr were transplanted into ischemic hindlimb mice model. Here we show that the activation of MAPK signaling pathways and the modulation of histone proteins such as Hp1-α, H3K27me3 and H3K4me3 in e/Entr contribute to the changes in chromatin configuration and reprogramming. Microarray data demonstrated that e/Entr highly expressed genes associated with ECs transcription factors and angiogenesis. In addition, the transplantation of e/Entr into hindlimb ischemia showed a high recovery of blood perfusion, limb salvage and e/Entr contributed to the formation of new vessels. In conclusion, the present study provided the first evidence that ultrasound reprogramming can induce postnatal cells to functional ECs. Therefore, our data suggest that physical stimulus-mediated reprogramming is a highly effective and safe strategy for the novel therapeutic alternatives.

Inhibition of STAT5A promotes osteogenesis by DLX5 regulation.

The regulation of osteogenesis is important for bone formation and fracture healing. Despite advances in understanding the molecular mechanisms of osteogenesis, crucial modulators in this process are not well-characterized. Here we demonstrate that suppression of signal transducer and activator of transcription 5A (STAT5A) activates distal-less homeobox 5 (DLX5) in human bone marrow-derived stromal cells (hBMSCs) and enhances osteogenesis in vitro and in vivo. We show that STAT5A negatively regulates expression of Dlx5 in vitro and that STAT5A deletion results in increased trabecular and cortical bone mass and bone mineral density in mice. Additionally, STAT5A deletion prevents age-related bone loss. In a murine fracture model, STAT5A deletion was found to significantly enhance bone remodeling by stimulating the formation of a fracture callus. Our findings indicate that STAT5A inhibition enhances bone formation by promoting osteogenesis of BMSCs.

Dual chemotactic factors-secreting human amniotic mesenchymal stem cells via TALEN-mediated gene editing enhanced angiogenesis.

BACKGROUND: Even though mesenchymal stem cells (MSCs) have angiogenic property, their cytokine secretory capacity is limited to treat ischemic vascular disorders. In present study, we produced genome-edited MSCs that secreted dual chemokine granulocyte chemotactic protein-2 (GCP-2) and stromal-derived factor-1α (SDF-1α) and determined their therapeutic potential in the context of experimental ischemia. METHODS: GCP-2 and SDF-1α genes were integrated into safe harbor site at the safe harbor genomic locus of amniotic mesenchymal stem cells (AMM) via transcription activator-like effector nucleases (TALEN). GCP-2 and SDF-1α gene-edited AMM (AMM/GS) were used for quantitative (q)-PCR, Matrigel tube formation, cell migration, Matrigel plug assays and in vivo therapeutic assays using hindlimb ischemia mouse model. RESULTS: AMM/GS-derived culture media (CM) induced significantly higher tube lengths and branching points as compared to AMM/S CM and AMM CM. Interestingly, Matrigel plug assays revealed that significantly higher levels of red blood cells were found in AMM/GS than AMM/S and AMM Matigel plugs and exhibited micro-vascular like formation. Cells was transplanted into ischemic mouse hindlimbs and compared with control groups. AMM/GS injection prevented limb loss and augmented blood perfusion, suggesting that enhances neovascularization in hindlimb ischemia. In addition, transplanted AMM/GS revealed high vasculogenic potential in vivo compared with transplanted AMM/S. CONCLUSION: Taken together, genome-edited MSCs that express dual chemokine GCP-2 and SDF-1α might be alternative therapeutic options for the treatment of ischemic vascular disease.

Development of Stabilized Growth Factor-Loaded Hyaluronate- Collagen Dressing (HCD) matrix for impaired wound healing.

BACKGROUND: Diabetes mellitus is a disease lack of insulin, which has severely delayed and impaired wound healing capacity. In the previous studies, various types of scaffolds and growth factors were used in impaired wound healing. However, there were several limitations to use them such as short half-life of growth factors in vivo and inadequate experimental conditions of wound-dressing material. Thus, our study aimed to determine the biocompatibility and stability of the matrix containing structurally stabilized epidermal growth factor (S-EGF) and basic fibroblast growth factor (S-bFGF). RESULTS AND DISCUSSION: We stabilized EGF and bFGF that are structurally more stable than existing EGF and bFGF. We developed biocompatible matrix using S-EGF, S-bFGF, and hyaluronate- collagen dressing (HCD) matrix. The developed matrix, S-EGF and S-bFGF loaded on HCD matrix, had no cytotoxicity, in vitro. Also, these matrixes had longer releasing period that result in enhancement of half-life. Finally, when these matrixes were applied on the wound of diabetic mice, there were no inflammatory responses, in vivo. Thus, our results demonstrate that these matrixes are biologically safe and biocompatible as wound-dressing material. CONCLUSIONS: Our stabilized EGF and bFGF was more stable than existing EGF and bFGF and the HCD matrix had the capacity to efficiently deliver growth factors. Thus, the S-EGF and S-bFGF loaded on HCD matrix had improved stability. Therefore, these matrixes may be suitable for impaired wound healing, resulting in application of clinical treatment.

Cell recruiting chemokine-loaded sprayable gelatin hydrogel dressings for diabetic wound healing.

UNLABELLED: In this study, we developed horseradish peroxidase (HRP)-catalyzed sprayable gelatin hydrogels (GH) as a bioactive wound dressing that can deliver cell-attracting chemotactic cytokines to the injured tissues for diabetic wound healing. We hypothesized that topical administration of chemokines using GH hydrogels might improve wound healing by inducing recruitment of the endogenous cells. Two types of chemokines (interleukin-8; IL-8, macrophage inflammatory protein-3α; MIP-3α) were simply loaded into GH hydrogels during in situ cross-linking, and then their wound-healing effects were evaluated in streptozotocin-induced diabetic mice. The incorporation of chemokines did not affect hydrogels properties including swelling ratio and mechanical stiffness, and the bioactivities of IL-8 and MIP-3α released from hydrogel matrices were stably maintained. In vivo transplantation of chemokine-loaded GH hydrogels facilitated cell infiltration into the wound area, and promoted wound healing with enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or the GH hydrogel alone. Based on our results, we suggest that cell-recruiting chemokine-loaded GH hydrogel dressing can serve as a delivery platform of various therapeutic proteins for wound healing applications. STATEMENT OF SIGNIFICANCE: Despite development of materials combined with therapeutic agents for diabetic wound treatment, impaired wound healing by insufficient chemotactic responses still remain as a significant problem. In this study, we have developed enzyme-catalyzed gelatin (GH) hydrogels as a sprayable dressing material that can deliver cell-attracting chemokines for diabetic wound healing. The chemotactic cytokines (IL-8 and MIP-3α) were simply loaded within hydrogel during in situ gelling, and wound healing efficacy of chemokine-loaded GH hydrogels was investigated in STZ-induced diabetic mouse model. These hydrogels significantly promoted wound-healing efficacy with faster wound closure, neovascularization, and thicker granulation. Therefore, we expect that HRP-catalyzed in situ forming GH hydrogels can serve as an injectable/sprayable carrier of various therapeutic agents for wound healing applications.

Synergistic Action of IL-8 and Bone Marrow Concentrate on Cartilage Regeneration Through Upregulation of Chondrogenic Transcription Factors.

The objective of this study was to determine whether a biphasic scaffold loaded with a combination of a chemokine and bone marrow concentrate (BMC) could improve tissue regeneration in knee articular cartilage of beagles with cylindrical osteochondral defects. For this investigation, an osteochondral defect (6 mm in diameter and 8 mm deep) was created in the weight-bearing articular surface of the femoral medial condyle in beagles. Bone marrow was aspirated from the posterior iliac crests of beagles to obtain mesenchymal stem cells (MSCs) for in vitro assay. Hematoxylin and eosin (HE), Masson's trichrome (MT), safranin O/fast green staining, and immunohistochemistry were performed for histological analysis. Quantitative real-time polymerase chain reaction was performed to understand the roles of BMC in chondrogenic differentiation of MSCs. At 12 weeks after transplantation of biphasic scaffolds, we observed that interleukin-8 (IL-8) or the combination of IL-8 and BMC induced massive bone regeneration compared to saline, BMC only, and MSCs. In gross appearance, the osteochondral defect site was nearly completely filled with repair tissue in the group that received the combination of IL-8 and BMC but not in the other groups. Moreover, histological analysis showed obvious differences in cartilage regeneration among groups. HE and MT staining showed that the cartilage defect sites of the group receiving the combination of IL-8 and BMC were regenerated with cartilage-like tissues showing chondrocyte morphology. Safranin O staining showed hyaline cartilage regeneration in the group receiving IL-8 and BMC, whereas fibrous-like tissues were formed in the other groups. Furthermore, immunostaining revealed the presence of type II collagen and aggrecan in regenerated cartilage tissue of the group receiving IL-8 and BMC, whereas regenerated cartilage tissues of the other groups weakly expressed type II collagen and aggrecan. These results indicate that the combination of a chemokine IL-8 and BMC has significant positive effects on osteochondral regeneration in a beagle model through enhancing expression of the chondrogenic transcription factors and markers such as Sox9 and type II collagen.